Organic cation compounds are moved across renal tubular epithelial cell membranes by multiple specific transporters, including a membrane potential-dependent transporter at the basolateral membrane and a proton/organic cation antiporter at the brush border membrane. Recently, the involvement of various transporters localized on the plasma membrane in the uptake system for nutrients and endogenous substances into cells and their transport mechanisms have been clarified (Tsuji and Tamai, Pharm. Res. 13 (1996), 963-977). These transporters recognize the structures of substances to be transported to selectively transport specific substances. Transporters that recognize the relatively wide range of structures may possibly recognize foreign substances such as drugs by mistake, and actively take in them into cells. It is believed that drugs permeate through the plasma membrane fundamentally by simple diffusion depending on their physicochemical properties such as molecular size, hydrophobicity, and hydrogen-binding capacity. Particularly, in the case of ionic drugs, only molecules in the non-dissociated form can permeate through the plasma membrane according to the pH partition hypothesis. However, it has become evident that a number of drugs penetrate through the cell membrane by a specific mechanism other than simple diffusion, that is, an active transport mediated by transporters, in organs that require efficient exchange of intracellular and extracellular substances, including, small intestine, uriniferous tubule, placenta, epithelial cells of choroid plexus, hepatocytes, and blood-brain barrier. Recently, cDNAs of several transporters have been cloned by the expression cloning method using Xenopus oocytes, a foreign gene expression system, and structural homology among them has been revealed (Fei et al., Nature 368 (1994), 563-566). For example, Gründemann et al. cloned an organic cation transporter, OCT1, which is assumed to be localized on a basement membrane, using the expression cloning method in 1994 (Gründemann et al., Nature 372 (1994), 549-552). Subsequently, OCT2 was identified by homology cloning based on the sequence of OCT1 (Okuda et al., Biochem. Biophys. Res. Commun. 224 (1996), 500-507). OCT1 and OCT2 show homology as high as 67% to each other (Gründemann et al., J. Biol. Chem. 272 (1997), 10408-10413). Both of them are intensely expressed in the kidney, but differ in the organ distribution; OCT1 is also expressed in the liver, colon, and small intestine, while OCT2 expression is specific to the kidney.
In 1996 Schömig and colleagues cloned a cDNA from cell line CAKI-1, a human kidney carcinoma cell line (DSMZ No. ACC 142) encoding a human putative integral membrane transport protein designated UT2h showing sequence similarity to organic cation transporter genes; see GenBank accession number Y09881.1 [GI:12053560]. More recently, Tamai et al., FEBS Lett. (1997), 107-111, reported on the cloning of a substantially identical cDNA encoding SLC22A4, which they named OCTN1. The deduced 551-amino acid protein contains 11 predicted transmembrane domains, a nucleotide-binding site motif, a motif conserved in sugar transporters, 4 potential N-glycosylation sites, and 5 potential protein kinase C phosphorylation sites. OCTN1 is 33% identical to OCT2 (SLC22A2) and 31% identical to OCT1 (SLC22A1). Recombinant OCTN1 expressed in mammalian cells has been reported to exhibit saturable uptake of the organic cation model substrate tetraethyl ammonium (TEA). TEA uptake was pH sensitive, with higher activity occurring at neutral and alkaline pH than at acidic pH. Depletion of cellular ATP decreased TEA uptake, indicating that OCTN1 transport occurs at least partially through an active process; the ATP dependence was greatest at acidic pH. Northern blot analysis detected a less than 2.5-kb OCTN1 transcript in several human fetal and adult tissues, with the highest expression levels found in fetal liver and adult kidney, trachea, and bone marrow. OCTN1 was also strongly expressed in several human cancer cell lines. Tamai et al. (1997) suggested that OCTN1 is a renal proton/organic cation antiporter functioning at the epithelial apical membrane. Similarly, international application WO99/13072 inter alia describes a human and a mouse gene, respectively, significantly homologous to organic cation transporter OCT1, encoding a transporter protein having organic cation transport activity for tetraethylammonium (TEA) and carnitine.
However, the biological implications of this organic cation transporter has not yet been elucidated.
International application WO03/054011 inter alia describes that genetic markers based on coding sequence mutations in the OCTN1 gene that significantly reduces its ability to transport the organic cation carnitine are associated with inflammatory bowel diseases such as severe, early-onset Crohn's Disease (CD). Recently, Tokuhiro et al., Nature Genetics 35 (2003), 341-348, reported on a significant association between SLC22A4 and rheumatoid arthritis, a common inflammatory disease with complex genetic components, in the Japanese population. They showed that expression of SLC22A4 is specific to hematologic and immunologic tissues and that SLC22A4 is also highly expressed in the inflammatory joints of mice with collagen-induced arthritis. A SNP affects the transcriptional efficiency of SLC22A4 in vitro, owing to an allelic difference in affinity to Runt-related transcription factor-1 (RUNX1), a transcriptional regulator in the hematopoietic system. A SNP in RUNX1 was also strongly associated with rheumatoid arthritis. However, the biological role of the SLC22A4 or OCTN1, in particular in view of its seemingly non-specificity for organic compounds remained unclear. Furthermore, the nature of the putative natural substrate molecules, if any, as well as the question whether generally an increase or decrease of transporter activity or selectively for only one such substrate molecule may be involved in disease processes remained still unknown. In addition, others published that the substrate specificity of OCTN1 was completely unresolved until now; see, e.g., di San Filippo et al., J. Biol. Chem. 278 (2003), 47776-47784. In fact, the OCTN1 remained the only organic cation transporter for which no endogenous compound has been found so far; see also review by Koepsell et al., Rev. Physiol. Biochem. Pharmacol. 150 (2003), 36-90, in particular table 3.
In view of the above, the technical problem underlying the present invention is to elucidate the substrate specificity of organic cation transporter OCTN1 (SLC22A4), which allows therapeutic intervention for disorders that are related to the malfunction or the lack of this transporter.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims, and described further below.